Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer.

AIMS: This study aims to explore the function and mechanism of G Protein-coupled receptor class C group 5 member A (GPRC5A) in docetaxel-resistance and liver metastasis of breast cancer. METHODS: Single-cell RNA transcriptomic analysis and bioinformatic analysis are used to screen relevant genes in breast cancer metastatic hepatic specimens. MeRIP, dual-luciferase analysis and bioinformation were used to detect m6A modulation. Mass spectrometry (MS), co-inmunoprecipitation (co-IP) and immunofluorescence colocalization were executed to explore the mechanism of GPRC5A in breast cancer cells. RESULT: GPRC5A was upregulated in triple-negative breast cancer (TNBC) and was associated with a poor prognosis. In vitro and in vivo experiments demonstrated that knockdown of GPRC5A alleviated metastasis and resistance to docetaxel in TNBC. Overexpression of GPRC5A had the opposite effects. The m6A methylation of GPRC5A mRNA was modulated by METTL3 and YTHDF1, which facilitates its translation. GPRC5A inhibited the ubiquitination-dependent degradation of LAMTOR1, resulting in the recruitment of mTORC1 to lysosomes and activating the mTORC1/p70s6k signaling pathway. CONCLUSION: METTL3/YTHDF1 axis up-regulates GPRC5A expression by m6A methylation. GPRC5A activates mTORC1/p70s6k signaling pathway by recruiting mTORC1 to lysosomes, consequently promotes docetaxel-resistance and liver metastasis.

Hsa_circ_0060467 promotes breast cancer liver metastasis by complexing with eIF4A3 and sponging miR-1205.

Breast cancer (BC) is the most common cancer and the top cause of female mortality worldwide. The prognosis for patients with breast cancer liver metastasis (BCLM) remains poor. Emerging studies suggest that circular RNAs (circRNAs) are associated with the progression of BC. Exploration of circRNAs presents a promising avenue for identifying metastasis-targeting agents and improving the prognosis of patients with BCLM. Microarray and bioinformatic analyses were used to analyze differentially expressed circRNAs between three pairs of BCLM and primary BC. The roles of hsa_circ_0060467 (circMYBL2) and its target gene E2F1 in BC cells were explored by multiple functional experiments. And xenograft mouse models and hepatic metastases of BC hemi-spleen models were used to illustrate the function of circMYBL2 in vivo. The intrinsic molecular mechanism involving circMYBL2 was confirmed by bioinformatics analyses, RIP assays, CHIRP assays, luciferase reporter assays, and rescue experiments. CircMYBL2 was overexpressed in BCLM tissues and BC cells. Functionally, circMYBL2 can facilitate the proliferation and liver metastasis of BC. Mechanistically, circMYBL2 upregulated the transcription factor E2F1 by sponging miR-1205 and complexing with eukaryotic translation initiation factor 4A3 (eIF4A3) and then facilitated the epithelial-mesenchymal transition (EMT) process in BC cells. Our findings showed that circMYBL2 promoted the tumorigenesis and aggressiveness of BC through the circMYBL2/miR-1205/E2F1 and circMYBL2/eIF4A3/E2F1 axes, which may provide a novel targeted therapy for patients with BCLM.

The Single-Cell Landscape of Intratumoral Heterogeneity and The Immunosuppressive Microenvironment in Liver and Brain Metastases of Breast Cancer.

Distant metastasis remains the major cause of morbidity for breast cancer. Individuals with liver or brain metastasis have an extremely poor prognosis and low response rates to anti-PD-1/L1 immune checkpoint therapy compared to those with metastasis at other sites. Therefore, it is urgent to investigate the underlying mechanism of anti-PD-1/L1 resistance and develop more effective immunotherapy strategies for these patients. Using single-cell RNA sequencing, a high-resolution map of the entire tumor ecosystem based on 44 473 cells from breast cancer liver and brain metastases is depicted. Identified by canonical markers and confirmed by multiplex immunofluorescent staining, the metastatic ecosystem features remarkable reprogramming of immunosuppressive cells such as FOXP3+ regulatory T cells, LAMP3+ tolerogenic dendritic cells, CCL18+ M2-like macrophages, RGS5+ cancer-associated fibroblasts, and LGALS1+ microglial cells. In addition, PD-1 and PD-L1/2 are barely expressed in CD8+ T cells and cancer/immune/stromal cells, respectively. Interactions of the immune checkpoint molecules LAG3-LGALS3 and TIGIT-NECTIN2 between CD8+ T cells and cancer/immune/stromal cells are found to play dominant roles in the immune escape. In summary, this study dissects the intratumoral heterogeneity and immunosuppressive microenvironment in liver and brain metastases of breast cancer for the first time, providing insights into the most appropriate immunotherapy strategies for these patients.

The FUS/circEZH2/KLF5/ feedback loop contributes to CXCR4-induced liver metastasis of breast cancer by enhancing epithelial-mesenchymal transition.

BACKGROUND: Metastasis of breast cancer have caused the majority of cancer-related death worldwide. The circRNAs are associated with tumorigenesis and metastasis in breast cancer according to recent research. However, the biological mechanism of circRNAs in liver metastatic breast cancer remains ambiguous yet. METHODS: Microarray analysis of three pairs of primary BC tissues and matched hepatic metastatic specimens identified circEZH2. We used RT-qPCR and FISH assays to confirm circEZH2 existence, characteristics, and expression. Both in vivo and in vitro, circEZH2 played an oncogenic role which promoted metastasis as well. A range of bioinformatic analysis, Western blot, RNA pull-down, RIP, ChIP, and animal experiments were used to define the feedback loop involving FUS, circEZH2, miR-217-5p, KLF5, FUS, CXCR4 as well as epithelial and mesenchymal transition. RESULTS: In our research, circEZH2 was proved to be upregulated in liver metastases in BC and predicted the worse prognosis in breast cancer patients. Overexpression of circEZH2 notably accentuated the vitality and invasion of BC cells, whereas knockdown of circEZH2 elicited the literally opposite effects. Besides, overexpressed circEZH2 promoted tumorigenesis and liver metastasis in vivo. Moreover, circEZH2 could adsorb miR-217-5p to upregulate KLF5 thus leading to activate FUS transcription which would facilitate the back-splicing program of circEZH2. Meanwhile, KLF5 could upregulated CXCR4 transcriptionally to accelerate epithelial and mesenchymal transition of breast cancer. CONCLUSIONS: Consequently, a novel feedback loop FUS/circEZH2/KLF5/CXCR4 was established while circEZH2 could be novel biomarker and potential target for BC patients' therapy.

Turning up a new pattern: Identification of cancer-associated fibroblast-related clusters in TNBC.

Growing evidence indicates a connection between cancer-associated fibroblasts (CAFs) and tumor microenvironment (TME) remodeling and tumor progression. Nevertheless, how patterns of CAFs impact TME and immunotherapy responsiveness in triple-negative breast cancer (TNBC) remains unclear. Here, we systematically investigate the relationship between TNBC progression and patterns of CAFs. By using unsupervised clustering methods in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset, we identified two distinct CAF-associated clusters that were related to clinical features, characteristics of TME, and prognosis of patients. Then, we established a CAF-related prognosis index (CPI) by the least absolute shrinkage and selection operator (LASSO)-Cox regression method. CPI showed prognostic accuracy in both training and validation cohorts (METABRIC, GSE96058, and GSE21653). Consequently, we constructed a nomogram with great predictive performance. Moreover, the CPI was verified to be correlated with the responsiveness of immunotherapy in three independent cohorts (GSE91061, GSE165252, and GSE173839). Taken together, the CPI might help us improve our recognition of the TME of TNBC, predict the prognosis of TNBC patients, and offer more immunotherapy strategies in the future.

The circROBO1/KLF5/FUS feedback loop regulates the liver metastasis of breast cancer by inhibiting the selective autophagy of afadin.

BACKGROUND: Metastasis causes the majority of cancer-related deaths worldwide. Increasing studies have revealed that circRNAs are associated with the carcinogenesis and metastasis of many cancers. Nevertheless, the biological mechanisms of circRNAs in breast cancer (BC) liver metastasis remain extremely ambiguous. METHODS: In this study, we identified circROBO1 from three pairs of primary BC and metastatic liver sites by RNA sequencing. FISH assays and RT-qPCR were conducted to validate the existence and expression of circROBO1. The oncogenic role of circROBO1 was demonstrated both in vitro and in vivo. Western blot, ChIP, RIP, RNA pull-down, and dual-luciferase reporter assays were used to confirm the interaction of the feedback loop among circROBO1, miR-217-5p, KLF5, and FUS. Meanwhile, the regulation of selective autophagy was investigated by immunofluorescence, CoIP, and western blot. RESULTS: In this study, upregulated expression of circROBO1 was found in BC-derived liver metastases and was correlated with poor prognosis. Knockdown of circROBO1 strikingly inhibited the proliferation, migration, and invasion of BC cells, whereas overexpression of circROBO1 showed the opposite effects. Moreover, overexpression of circROBO1 promoted tumor growth and liver metastasis in vivo. Further research revealed that circROBO1 could upregulate KLF5 by sponging miR-217-5p, allowing KLF5 to activate the transcription of FUS, which would promote the back splicing of circROBO1. Therefore, a positive feedback loop comprising circROBO1/KLF5/FUS was formed. More importantly, we found that circROBO1 inhibited selective autophagy of afadin by upregulating KLF5. CONCLUSIONS: Our results demonstrated that circROBO1 facilitates the carcinogenesis and liver metastasis of BC through the circROBO1/KLF5/FUS feedback loop, which inhibits the selective autophagy of afadin by suppressing the transcription of BECN1. Therefore, circROBO1 could be used not only as a potential prognostic marker but also as a therapeutic target in BC.

A Novel Pyroptosis-Associated Long Non-coding RNA Signature Predicts Prognosis and Tumor Immune Microenvironment of Patients With Breast Cancer.

Background: Pyroptosis, a kind of programmed cell death characterized by the rupture of cell membranes and the release of inflammatory substances, plays an important role in the occurrence and development of cancer. However, few studies focus on the pyroptosis-associated long non-coding RNAs (lncRNAs) in breast cancer (BC). The prognostic value of pyroptosis-associated lncRNAs and their relationship with tumor microenvironment (TME) in BC remain unclear. The purpose of this study was to explore the prognostic role of pyroptosis-associated lncRNAs and their relationship with TME in BC. Methods: The transcriptome data and clinical data of female BC patients were downloaded from The Cancer Genome Atlas (TCGA) database. A total of 937 patients were randomly assigned to either training set or validation set. A pyroptosis-associated lncRNA signature was constructed in the training set and verified in the validation set. Functional analysis and immune microenvironment analysis related to pyroptosis-associated lncRNAs were performed. A nomogram based on the risk score and clinical characteristics was established. Results: A 9-pyroptosis-associated lncRNA signature was constructed to separate BC patients into two risk groups. High-risk patients had poorer prognosis than low-risk patients. The risk score was proven to be an independent prognostic factor by multivariate Cox regression analysis. Function analysis and immune microenvironment analysis showed that low-risk BC tended to be an immunologically "hot" tumor. A nomogram was constructed with risk score and clinical characteristics. Receiver operating characteristic curve (ROC) analysis demonstrated credible predictive power of the nomogram. The area under time-dependent ROC curve (AUC) reached 0.880 at 1 year, 0.804 at 3 years, and 0.769 at 5 years in the training set, and 0.799 at 1 year, 0.794 at 3 years, and 0.728 at 5 years in the validation set. Conclusion: We identified a novel pyroptosis-associated lncRNA signature that was an independent prognostic indicator for BC patients. Pyroptosis-associated lncRNAs had potential relationship with the immune microenvironment and might be therapeutic targets for BC patients.

A Systemic Inflammation Response Score for Prognostic Prediction of Breast Cancer Patients Undergoing Surgery.

Background: Systemic inflammatory response is related to the occurrence, progression, and prognosis of cancers. In this research, a novel systemic inflammation response score (SIRS) was calculated, and its prognostic value for postoperative stage I-III breast cancer (BC) patients was analyzed. Methods: 1583 BC patients were included in this research. Patients were randomly divided into a training cohort (n = 1187) and validation cohort (n = 396). SIRS was established in the training cohort based on independent prognostic hematological indicator, its relationship between prognosis and clinical features was analyzed. Then, a nomogram consisted of SIRS and clinical features was established, its performance was examined by calibration plots and receiver operating characteristic curve analysis. Results: The SIRS was an independent prognostic indicator for BC patients, and a high-SIRS was related to multifocality, advanced N stage, and worse prognosis. Incorporating SIRS into a nomogram could accurately predict the prognosis of BC patients, the results of receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) of nomogram was up to 0.806 in training cohort and 0.905 in the validation cohort. Conclusion: SIRS was associated with the prognosis of patients with breast cancer. Nomogram based on SIRS can accurately predict the prognosis of breast cancer patients.

A Novel Platelet-Related Gene Signature for Predicting the Prognosis of Triple-Negative Breast Cancer.

Regarded as the most invasive subtype, triple-negative breast cancer (TNBC) lacks the expression of estrogen receptors (ERs), progesterone receptors (PRs), and human epidermal growth factor receptor 2 (HER2) proteins. Platelets have recently been shown to be associated with metastasis of malignant tumors. Nevertheless, the status of platelet-related genes in TNBC and their correlation with patient prognosis remain unknown. In this study, the expression and variation levels of platelet-related genes were identified and patients with TNBC were divided into three subtypes. We collected cohorts from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. By applying the least absolute shrinkage and selection operator (LASSO) Cox regression method, we constructed a seven-gene signature which classified the two cohorts of patients with TNBC into low- or high-risk groups. Patients in the high-risk group were more likely to have lower survival rates than those in the low-risk group. The risk score, incorporated with the clinical features, was confirmed as an independent factor for predicting the overall survival (OS) time. Functional enrichment analyses revealed the involvement of a variety of vital biological processes and classical cancer-related pathways that could be important to the ultimate prognosis of TNBC. We then built a nomogram that performed well. Moreover, we tested the model in other cohorts and obtained positive outcomes. In conclusion, platelet-related genes were closely related to TNBC, and this novel signature could serve as a tool for the assessment of clinical prognosis.

LGR6 Promotes Tumor Proliferation and Metastasis through Wnt/ß-Catenin Signaling in Triple-Negative Breast Cancer.

Leucine-rich-repeat-containing G protein-coupled receptor 6 (LGR6) has been identified as the stem cell marker in multiple normal tissues and malignancies. Previous studies implicated paradoxical functions of LGR6 as a tumor-suppressor gene or oncogene given to the specific context. To explore the exact role of LGR6 in triple-negative breast cancer (TNBC) that never has been studied before, in this study, we assessed LGR6 expression levels by RT-PCR and immunohistochemistry. LGR6 stable expressing/silenced cells were established, and functional assays on tumor proliferation, as well as metastasis, were conducted both in vitro and in vivo. Here, we found that LGR6 was overexpressed in TNBC, which correlated with poor disease-free and overall survivals. Functional assays both in vitro and in vivo showed that LGR6 promotes tumor proliferation and metastasis. LGR6 also increased the ability of tumor spheroid formation. Underlying mechanism exploration further revealed that the oncogenic role of LGR6 might be associated with the Wnt/ß-catenin pathway. In conclusion, our findings first proved that LGR6 acts as an oncogene in (TNBC), indicating that LGR6 might be a potential therapeutic target for TNBC treatment.

miR-200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer.

Resistance to trastuzumab remains a major obstacle in HER2-overexpressing breast cancer treatment. miR-200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR-200c contributes to trastuzumab resistance and stemness maintenance in HER2-overexpressing breast cancer. In this study, we used HER2-positive SKBR3, HER2-negative MCF-7, and their CD44+ CD24- phenotype mammospheres SKBR3-S and MCF-7-S to verify. Our results demonstrated that miR-200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR-200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44+ CD24- phenotype mammospheres in SKBR3-S. Combining miR-200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3-S. Overexpression of miR-200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR-200c also improved the malignant progression of SKBR3-S and SKBR3 in vivo. miR-200c plays an important role in the maintenance of the CSC-like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.

circFBXW7 Inhibits Malignant Progression by Sponging miR-197-3p and Encoding a 185-aa Protein in Triple-Negative Breast Cancer.

Accumulating evidence indicates that circular RNAs (circRNAs) are vital regulators of various biological functions involved in the progression of multiple cancers. Circular F-box and WD repeat domain containing 7 (circFBXW7) (hsa_circ_0001451) has been reported to act as a tumor suppressor by encoding a novel protein in glioma; however, its functions and mechanisms in triple-negative breast cancer (TNBC) remain elusive. In the current study, we validated by qRT-PCR that circFBXW7 was downregulated in TNBC cell lines and found that low expression of circFBXW7 was associated with poorer clinical outcomes. circFBXW7 expression was negatively correlated with tumor size and lymph node metastasis, and it was an independent prognostic factor for TNBC patients. We performed cell proliferation, colony formation, transwell, wound-healing, and mouse xenograft assays to confirm the functions of circFBXW7. Overexpression of circFBXW7 obviously inhibited cell proliferation, migration, and tumor growth in both in vitro and in vivo assays. Luciferase reporter assays and RNA immunoprecipitation assays revealed that circFBXW7 serves as a sponge of miR-197-3p and suppresses TNBC growth and metastasis by upregulating FBXW7 expression. In addition, the FBXW7-185aa protein encoded by circFBXW7 inhibited the proliferation and migration abilities of TNBC cells by increasing the abundance of FBXW7 and inducing c-Myc degradation. In summary, our research demonstrated that circFBXW7 sponges miR-197-3p and encodes the FBXW7-185aa protein to suppress TNBC progression through upregulating FBXW7 expression. Thus, circFBXW7 may act as a therapeutic target and prognostic biomarker for TNBC.

CircPLK1 sponges miR-296-5p to facilitate triple-negative breast cancer progression.

Aim: To investigate the role of circRNAs in triple-negative breast cancer (TNBC) and the underlying mechanisms. Materials & methods: We performed circRNA microarrays to explore the expression profiles of TNBC cell lines. Experiments in vitro and in vivo were conducted to explore the effects of circPLK1 on tumor proliferation and metastasis as well as the interaction between circPLK1, miR-296-5p and PLK1 in TNBC. Results & conclusion: CircPLK1 was significantly upregulated in TNBC and associated with poor survivals. CircPLK1 knockdown inhibited cell growth and invasion in vitro as well as tumor occurrence and metastasis in vivo. CircPLK1-miR-296-5p-PLK1 axis regulates tumor progression by ceRNA mechanism in TNBC, indicating that circPLK1 may serve as a prognostic factor and novel therapeutic target for TNBC.

MALAT1 and BACH1 are prognostic biomarkers for triple-negative breast cancer.

AIMS: The purpose of this study was to investigate the potential correlation between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the transcription factor BTB and CNC homology 1 (BACH1) and their clinicopathological significance in triple-negative breast cancer (TNBC). SUBJECTS AND METHODS: MALAT1 and BACH1 were detected by immunohistochemistry using TNBC tissue microarrays of 240 patients. The association between MALAT1 and BACH1 expression levels was statistically analyzed. Moreover, the prognostic roles as well as clinical and pathological significance of MALAT1 and BACH1 expression in TNBC were determined. STATISTICAL ANALYSIS USED: Two-tailed Pearson correlation was used to examine the correlation of BACH1 and MALA1 expression. Comparisons of clinicopathological variables between different BACH1 and MALA1 expression groups were performed using χ2 tests. Overall survival (OS) and disease-free survival (DFS) curves were plotted with the Kaplan-Meier method and the differences in OS and DFS between three groups were compared by the log-rank test. Multiple comparisons were performed using χ2 tests for subsequent individual group comparisons. RESULTS: MALAT1 and BACH1 expression was significantly correlated with tumor-node-metastasis stage, distant metastasis, pathological stage, and survival outcomes of patients. Patients with high MALAT1 and BACH1 expression exhibited shorter overall survival and disease-free survival. CONCLUSIONS: These findings provide further insight into the expression pattern of MALAT1 and BACH1 in TNBC and suggest them as prognostic biomarkers for TNBC.